Project Summary/Abstract Oxidative stress is induced by proteasome inhibitors (PIs) and contributes to the robust cytotoxic effects of these drugs in Multiple Myeloma (MM) cells. Likewise, dysregulation or alterations in redox signaling have been reported in cells that have acquired resistance to PIs. This suggests that redox changes contribute to PI resistance and that specific redox pathway regulators are promising therapeutic targets for PI refractory MM. PI resistance is a significant obstacle in the treatment of MM, and new therapeutic approaches that specifically target molecular mechanisms that drive resistance are needed to maximize the potential of PIs and produce durable response and ultimately cures. Preliminary studies using isogenic pairs of PI sensitive and resistant MM cells identified a set of redox and bioenergetics changes associated with PI resistance. These changes include increased mitochondrial respiration and reactive oxygen species (ROS) production, along with increased protection from PI-induced oxidative damage to macromolecules. This combination of observations strongly implicates the mitochondrial redox protein Nicotinamide Nucleotide Transhydrogenase (NNT), which uses the mitochondrial membrane potential to generate reducing equivalents for anti-oxidant protection in the mitochondria. Parallel drug discovery studies identified a promising redox-modulating compound, E61, which synergistically overcomes PI resistance in MM cells while protecting normal primary cells from PI-induced cell death. The specific goals of the proposed project are (1) to confirm the role of NNT as a mediator of PI resistance in MM cells, (2) to identify the direct redox binding target of developmental compound E61, and (3) to establish the preclinical anti-MM potential of compound E61 using a mouse model of myeloma bone disease. A combination of cutting edge techniques and tools will be incorporated into the experimental plan. Examples include the use of CRISPR/Cas gene editing technology to stably silence the NNT gene in PI resistant cells, the use of click chemistry coupled with mass spectrometry to immobilize and identify putative E61 binding targets, and the use of a NOD-SCID IL2Rgammanull (NSG) mouse model of MM, in which cells are injected systemically and form bone lesions that resemble the human MM pathological condition. Through the use of these innovative tools directed toward the specific aims of the project, this study will clarify the role of redox in the acquisition of PI resistance and deliver a promising new compound for the treatment of refractory MM.